This invention relates to a process for preparing functional sugar polymers and an apparatus useful for the synthesis of such sugar polymers.
Sugar polymers produced by microorganisms and plants, mainly straight and branched glucans and fructans, have a long history of applicability in food products as gums, fillers, bulk sources, and other similar usage. In addition to their uses in foods some of these sugars have long been purported to impart health benefits. See, McAuliffe, J., Hindsgaul, O.; xe2x80x9cCarbohydrate Drugsxe2x80x94An Ongoing Challengexe2x80x9d, Chemistry and Industry, Mar. 3, 1997. More recently a growing number of these polymers (whether of plant, microorganism or mammalian origin), especially those having unique branching or containing other sugars, such as fucose, galactose, sugar amines, or sialic acid, have been cited as imparting a variety of health benefits (see, for example, U.S. Pat. No. 5,514,660). While a great deal is known concerning the specific use and functionality of many of the significant natural sugar polymers, the complexity of these compounds and the resultant difficulty and high cost of preparing structures which may not exist in nature has limited the knowledge of general structure/function or health benefit relationships. Sugar polymers are generally isolated from plants or microorganisms by extraction and purified by a variety of techniques. Significant work has been done to increase the availability and functionality and to decrease the cost of these polymers. This work has centered in the following areas:
Better sources of the plant sugars have been found through the discovery of new species, selective breeding, and genetic manipulation.
Improved processes to extract the sugar polymers have been developed.
The sugar polymers have been separated into discrete chain length fractions and/or xe2x80x9crefinedxe2x80x9d to a tighter specification.
Simple chemical modifications to some plant sugars have been developed.
Work is ongoing and accelerating in all of these areas. One important area with significant on-going effort is the genetic modification of organisms to improve the production of the existing sugars or to make new sugars.
Methods to obtain sugar polymers without using plants are also known. Both chemical and enzymatic synthesis have been developed (see, for example, Whitesdie, G. M., et al.; xe2x80x9cEnzyme-Catalyzed Synthesis of Carbohydratesxe2x80x9d, Tetrahedron, 45(17), pp. 5365-5422, 1989). Chemical synthesis, while possible is hindered by three factors. First, each of the monomeric units has multiple reaction sites. In normal chemical synthesis multiple reactive sites always add complexity but with carbohydrates this complexity is extremely significant. A polymer with just four sugar xe2x80x9cmonomerxe2x80x9d units, can be assembled in xcx9c270,000 possible ways. This complexity can be overcome by selective blocking and de-blocking of reactive sites but only at the expense of low yield and high processing cost. Second, the inter-sugar bonds are very labile under typical processing conditions again limiting synthetic choices and, decreasing yields. Finally, many chemical reactions are not stereo-specific, creating a separation problem and further yield reductions. The net results are high cost and general lack of availability. Chemical synthesis is clearly appropriate for production of sugar polymers only when the product carries a very high value.
Processes that use enzymes as selective catalysts offer more promise for the synthetic production of sugar polymers. Recent advances in the identification and production of these enzymes, as well as systems in which to accomplish multi-step coupling reactions have reduced the cost of producing sugar polymers by many orders of magnitude. These techniques are applicable to both xe2x80x9cnaturalxe2x80x9d polymers and novel polymers based on coupling xe2x80x9cnaturalxe2x80x9d carbohydrate sub-units in new ways. Enzymes have been identified which allow coupling of inexpensive starting materials, such as sucrose, making this type of process potentially very inexpensive. Enzyme based production provides for both coupling and stereo selectivity which minimizes purification costs (see U.S. Pat. No. 5,288,637, Roth, S., xe2x80x9cApparatus for the Synthesis of Saccharide Compositionsxe2x80x9d; U.S. Pat. No. 5,180,674, Roth, S., xe2x80x9cSaccharide Compositions, Methods, and Apparatus for Their Synthesisxe2x80x9d; PCT/US92/10891, Roth, S., xe2x80x9cA Method for Obtaining Glycosyltransferasesxe2x80x9d; PCT/US95/12317, Gotschlich, E. C., xe2x80x9cGlycosyltransferases for Biosynthesis of Oligosaccharides and Genes for Encoding Themxe2x80x9d; PCT/US94/07807, Roth, S., xe2x80x9cA Method for Synthesizing Saccharide Compositionsxe2x80x9d). While these techniques allow great control of the coupling reactions, control of the chain length or branch distribution in polymers is not addressed.
An example of a sugar polymer is inulin. This polymer consists of fructose units, joined linearly in a xcex2-[2-xe2x86x921] fashion to a terminal xcex1-D-glucopyranosyl unit. In many plants inulin is a major energy storage carbohydrate. It is highly regarded as a naturally low calorie foodstuff, which can be used to provide bulk to high intensity sweeteners, as a soluble fiber source, as a fat replacer, and as a promoter of bifidus bacteria in the digestive tract. Inulin is commonly produced by extraction from plants, notably chicory and Jerusalem artichoke. The material extracted from these plants contains polymers with from 1 to over 60 fructose units attached to a terminal glucose unit. Polymer distributions vary with the plant source, the planting location, and the harvest time. While the crude extract has application, product with 3 to 10 fructose units provides the most xe2x80x9csucrose-likexe2x80x9d performance. These polymers make ideal bulking agents and fiber sources as they have low caloric density, are bland, and have many of the functional attributes of sucrose. Shorter polymers, while functional, provide a disproportionate increase in calories and sweetness. Longer polymers are less water-soluble and have good fat replacer properties but decreased. sucrose replacement functionality.
Several approaches have been used to provide improved functionality and consistent inulin product over a growing season. The first approach involves using a plant source that contains relatively long chain inulin and using selective enzymes to reduce the chain size in a controlled manner. EP 440074 relates to the selective hydrolysis of long-chain inulins. This allows some control over seasonal variations at the cost of adding a process step and does not help to eliminate the high calorie, short chain products. Another approach is to fractionate the crude inulin into cuts with different functionality. While this solves the functionality and seasonal variation problems, it does so at the expense of creating by-products. This approach also provides for removal of short chain polymers. Since it only involves fractionation it adds no xe2x80x9cforeignxe2x80x9d substances to the product. The two approaches can be combined in several ways to optimize functional product recovery. In both of these cases the agricultural source is grown specifically for the inulin product with all the risks associated with any agricultural program. These approaches also present the problem of disposal of a large mass of unused vegetable material. Finally, except for very high use sugars such as sucrose, it is difficult to generate any economies of scale when growing a plant for a specific product.
Another method to produce inulin fructooligosaccharides is to build up the fructose polymer from an inexpensive fructose source, such as sucrose, using coupling enzymes. (See Jong Won Yun, xe2x80x9cFructooligosaccharidesxe2x80x94Occurrence, Preparation, and Applicationxe2x80x9d, Enzyme and Microbial Technology 19:107-117, 1996, and U.S. Pat. No. 4,681,771). This approach suffers from inhibition of the enzymatic reaction by a by-product of the coupling, glucose, which limits the conversion of the sucrose feed, and the length of the chains produced. These inulin fructooligosaccharide compositions, commonly have an average chain length of under 5 fructose units.
Enzyme catalyzed reactions in biological systems are regulated by the inhibitory effects on the enzyme by the accumulation of by-products. This is used to the organism""s advantage to prevent the accumulation of unused reaction products. Where the enzyme is used as part of a cascade of reactions, the product of an enzyme is removed by the actions of the next enzyme in the cascade. When enzymes are used in man-made reactors, alternative methods must be employed. One approach used to remove the inhibition is to scavenge the glucose that inhibits the forward reaction by converting it to another by-product. While this may improve yield, it does little to improve selectivity of chain length. Other techniques that remove glucose, such as permeation though a selective membrane, are also possible, but again only results in improved yield and fail to produce the desired polymers. Another refinement, commonly used in multi-step enzymatic reactors, is to use a series of reactors with inter-reactor removal of by-products, and if desirable, addition of feed materials. For a significant increase in system complexity this type of system can result in improved yield. While it also allows some control over the average of the polymer size, it offers little control of the chain length distribution.
Chain length distribution cannot be predicted using simple Michaelis-Menten kinetics (F. Ouarne and A. Guibert, xe2x80x9cFructooligosaccharides Enzymic Synthesis from Sucrose.xe2x80x9d Zuckerindustrie (Berlin) (1995), 120, pp 793-798).
Chain elongation of the fructooligosaccharide chains by sequential transfer of fructose from sucrose by A. niger fructosyltransferase is reported by M. Hirayama and H. Hidaka, (Chapter 9, xe2x80x9cProduction and Utilization of Microbial Fructansxe2x80x9d in Science and Technology of Fructans, CRC Press, 1993):
2 GFxe2x86x92G+GF2 
GF+GF2xe2x86x92G+GF3 
GF+GF3xe2x86x92G+GF4, etc.
As used above, the term GF refers to sucrose and the term GFn refers to a xcex2-2,1-linked fructose oligosaccharide which is linked via its reducing end to an alpha-D-glucopyranosyl moiety.
A different model for the elaboration of fructooligosaccharide chains is proposed in K. J. Duan, J. S. Chen and D. C. Cheu., xe2x80x9cKinetic Studies and a Mathematical Model for Enzymatic Production of Fructooligosaccharides from Sucrose; Fructooligosaccharide Sweetener Production Using Aspergillus japonicus beta-D-fructofuranosidase.xe2x80x9d Enzyme Microb. Technol., 4, 334-339 (1994)). Two like-sized chains react with the enzyme to give a shorter and a longer chain, and a hydrolysis step that liberated fructose from the tetrasaccharide GF3:
2 GFnxe2x86x92GFnxe2x88x921+GFn+1 for n=1 to 3
GF3xe2x86x92F+GF2 
F. Ouarne and A. Guibert (xe2x80x9cFructooligosaccharides: Enzymic Synthesis from Sucrose.xe2x80x9d Zuckerindustrie, (Berlin), 120, 793-798, (1995)) discloses a mathematical model based on the reaction pattern above, and added the transfer of fructose from the trisaccharide GF2 to glucose to produce two moles of sucrose and the hydrolysis reaction of sucrose to its component monosaccharides:
G+GF2xe2x86x922 GF
GFxe2x86x92F+G
The model described in Zuckerindustrie showed good agreement with observed experimental values in the production of the various components in the reaction mixture, but do not preclude the stepwise elongation of chains by sequential transfer of fructose from sucrose as a possible contributing mechanism.
The model described in Zuckerindustrie is based on well documented enzyme kinetics and predicts the performance reported for the production of fructooligosaccharides. A quick analysis of the model will reveal why these chain length distributions are obtained. The rate of formation of GFn+1 is dependent on the concentration of GFn. In both batch and plug flow reaction systems, this relation sets the type of distributions which will be seen. Pushing the reaction harder through increased concentrations, catalyst levels, or residence time doesn""t affect the relative distribution, only its position. In the case of fructose polymers, this means that by increasing the extent of the reaction you can shift the product distribution from mostly GF2 with progressively smaller amounts of GF3, GF4, etc., to mostly GF3 with progressively smaller amounts of longer chains by pushing the reaction harder. Plug flow type reactor configurations, or multiple back-mixed reactors used in series increase the amounts of high polymers but again cannot impact the basic relation. If the polymerization reaction is considered essentially irreversible, the average length is clearly limited by these relations. When the GF supply is exhausted, no further growth will take place. If more sucrose is added, the reaction will go forward through GF2. Assuming the problem of glucose inhibition can be overcome, this can be improved on by adding sucrose between the stages of a series of plug flow or back-mixed reactors. While this is feasible, it would certainly add significant cost and complexity to the system to achieve any meaningful improvements. Where reversible polymerization is possible, it may be possible to push the reaction further, but a new set of limitations would exist. In this case, an equilibrium level of products would be achieved, assuming sufficient residence time was given. Adding more sucrose would increase the total material produced but not the ratios of product. Likewise, other reactor configurations would also not change the final reaction mix.
The invention relates to a process for preparing a straight or branched sugar polymer of a desired chain length, preferably four or more units, comprising:
a) transferring a monosaccharide or oligosaccharide residue from a carbohydrate donor to an acceptor by means of selective catalytic transfer, preferably by reacting with an enzyme;
b) optionally, removing by-products from step a) which may inhibit yield or selectivity;
c) separating polymers which have not achieved the desired chain length; and
d) recycling the polymers which have not achieved the desired chain length.
In another aspect, the invention relates to an apparatus for producing straight or branched sugar polymers of a desired chain length comprising:
a) a reactor in which a monosaccharide or oligosaccharide residue is transferred by selective catalytic transfer from a carbohydrate donor to an acceptor;
b) means for removing by-products from the reaction of step a);
c) means for separating polymers which have not achieved the desired chain length; and
d) means for recycling the polymer chains which have not achieved the desired chain length back to the reactor.
We have discovered that by combining a means for separating underdeveloped polymers and by-products, for example, by selective membrane, such as nano-filters, with an enzyme based reactor you not only increase yield but also tailor the resultant product to a highly specific range. Using membranes in conjunction with the enzyme system provides two key benefits. First a membrane is used to remove the by-product, glucose, without converting it to another entity. This allows the reaction to proceed to high yields without adding reaction steps to the process. A second membrane is added to remove chains of the desired length from the system while recycling shorter (underdeveloped) chains back for further growth.
This novel combination results in a system that out performs the conventional approaches by a significant margin. Again, looking to the kinetic model, the reasons that this novel combination gives superior performance is clear. When a separation system is added as described herein the entire dynamic is changed. For example, GF3""s produced are not removed from the system but are the source to produce GF4""s which are taken as product. The concentrations in the reactor necessary to produce longer chains can be present without impacting the product distribution. The fundamental relations in the reactor remain, but do not effect the product mix. This is true regardless of whether the reaction is considered reversible or irreversible. Through judicious choice of membrane, any average polymer chain length distribution can be achieved while maintaining virtually 100% yield.